Acute lung injury (ALI), including hyperoxic lung injury, is a syndrome of great public health importance. Although the pathogenesis of ALI remains unclear, an imbalance between production and elimination of reactive oxygen and nitrogen species is well accepted as a contributing factor. Pulmonary endothelium is the locus of early structural and functional changes in hyperoxic lung injury and apoptosis of pulmonary endothelium is a contributing factor to genesis and maintenance of the injury. Nonetheless, the early molecular pathways that account for hyperoxic induced pulmonary endothelial cell apoptosis are unknown and therapies for ALI remain entirely palliative. We have recently shown that upon interaction with anionic phospholipids, particularly mitochondria-specific cardiolipin (CL), cytochrome c (cyt c) loses its tertiary structure and its peroxidase activity dramatically increases. CL-induced peroxidase activity of cyt c has been found to be important for selective CL oxidation in cells undergoing programmed death. During apoptosis, the peroxidase activity and the fraction of CL-bound cyt c markedly increase, suggesting that CL acts as a switch to regulate cyt c's mitochondrial functions. In preliminary data, using ion trap electrospray ionization mass spectrometry based lipidomics for the first time in lung, we show that selective oxidation of CL is an early event in hyperoxic lung and also occurs in simple models (e.g. LPS) of apoptosis in cultured pulmonary endothelial cells. Furthermore we have designed an antioxidant/electron scavenger (XJB-5-131;an hemigramicidin-4-amino-2,2,6,6- tetramethylpiperidine-N-oxyl) and a peroxidase-activatable nitric oxide donor (HVTP, 2- hydroxylamino-vinyl-triphenyl-phosphonium) that are specifically targeted to mitochondria and show that they can effectively inhibit apoptosis in a variety of cell types and ameliorate acute organ dysfunction in vivo. Accordingly, the SPECIFIC AIMS of this proposal are to determine: 1) the molecular role of CL-induced peroxidase activity of cytochrome c in pulmonary endothelial cell apoptosis in hyperoxic mouse lung;2) the molecular role of oxidized anionic phospholipids in the signal transduction pathway of pulmonary endothelial cell apoptosis including scavenging of apoptotic pulmonary endothelium by macrophages;3) the therapeutic potential of XJB-5-131 and HVTP, two mitochondria-targeted antioxidants/inhibitors of cyt c/CL peroxidase, in preventing pulmonary endothelial cell CL oxidation, apoptosis and favorably affecting the course of hyperoxic lung injury. PUBLIC HEALTH RELEVANCE: While antioxidants have been long viewed as potentially effective for therapeutic interventions, elucidation of specific mechanisms through which cyt c and CL oxidation products link free radical production with early apoptotic events in mitochondria will contribute to a roadmap to new effective drug discovery targets.